Abstract Restricted amounts of biological samples that can be collected from pediatric patients for the study of immune function are often insuf?cient for conventional assays. Development of novel, multi-parameter sample sparing assays is necessary for advancing the fundamental understanding of the immune system. In this proposal, we outline three aims which detail the addition and integration of new biological readouts to an existing micro?uidic technology and the use of this expanded platform to investigate the mechanisms for how allergies and asthma develop in children. The kit-on-a-lid- assay (KOALA) is an easy to use self-contained micro?uidic platform which is capable of isolating neutrophils from whole blood, and assessing their chemotactic abilities. In Aim 1 we propose to adapt and validate assays for measuring neutrophil ROS production, NETosis, phagocytosis, cytokine production, and gene expression (RT- qPCR) into KOALA. We also plan to modify the cell capture speci?city of KOALA to allow us to isolate T cells from whole blood and assess their cytokine production and gene expression (RT-qPCR). The last component of Aim 1 is to test and compare a new, less invasive blood draw method called Hemolink? against traditional blood draw methods in terms of immune cell phenotype. In Aim 2 we propose to use a single-cell real-time non-invasive label-free microscopy method called optical metabolic imaging (OMI) to identify single-cell heterogeneity in activation within T cell and neutrophil populations. OMI is compatible with the other endpoints and can be run simultaneously. In Aim 3 will apply our multiplexed KOALA assays to blood samples from a cohort of children with allergic disease and matched healthy controls from the WISC cohort (an ongoing study at UW that includes farm and non-farm children). We will compare our neutrophil and T cell function and phenotype results from these children with the extensive measurements being done as part of the WISC cohort study. In Aim 3 we will also extend these analyses to a group of infants from the CREW study (a new study at UW that aims to identify early life genetic and environmental risk factors for allergic diseases and asthma) using the HemoLink? device to draw blood with minimal discomfort and pro?le type 2 T cell function (T cells differentiated to secrete IL-4, IL-5, IL-13 and other type 2 cytokines) using KOALA. If successful in our aims, we will have created a miniaturized, highly multiplexed, self-contained micro?uidic platform that is capable of measuring biological markers related to immune status and function. This system will have then been validated in pediatric subjects and ready for transition into clinical research.